This invention relates to yeast artificial chromosomes and their manipulation and transfer into cells and animals, to exploit the control of gene expression, and also to the resulting cells and animals.
The ability to transform suitable hosts with foreign DNA, and thus to express gene products not normally produced by the host, is an important goal of biotechnological research. Microorganisms can be used to produce desired proteins, while higher animals having desirable characteristics have also been produced. For example, EP-A-0264166, WO-A-8800239, WO-A-8801648, WO-A-9005188 and WO-A-9211358 describe the use of lactating animals to express foreign proteins which are produced in the milk and can be isolated therefrom; this provides a very satisfactory, controlled source of pure protein.
Techniques to transfer cloned DNA into mammalian cells and transgenic animals have greatly facilitated the study of gene regulation and expression. Gene transfection experiments have also highlighted the fact that the limited size of many cloned DNA molecules prevents the efficient use of the numerous distant regulatory sequences believed to control expression.
More particularly, in order to investigate regulation of complex loci and chromosomal domains harbouring clusters of genes, it is essential to introduce very large pieces of DNA into cells and animals. The conventional approaches used in transgenic animal technology have limitations, making it difficult to introduce DNA fragments which are greater than 100 kb; see Brxc3xcggemann et al, Eur. J. Immunol. 21:1323-1326 (1991). For example, while germ line-dependent genomic imprinting may affect large areas in which a number of genes may be similarly regulated, there is no efficient method to study such xe2x80x9cimprintedxe2x80x9d domains. A satisfactory technique for introducing large DNA fragments would allow progress, and also facilitate the analysis of other complex loci such as the T-complex (harbouring specific deletions) for which a number of genes have been mapped which are crucial for mammalian development. In order to obtain a better understanding of the regulation of eukaryotic genes, it would be desirable to express these genes in their authentic genomic context after cloning and re-introduction into cells.
The expression of mammalian genes is controlled at various levels: the genomic context (influence of neighbouring genes), regulatory elements proximal to the exons (e.g. promoters), regulatory sequences downstream of the termination codon (polyadenylation site) and regulatory motifs further away from coding sequences (e.g. enhancers). For immunoglobulin genes, it has been shown that expression of transgenes is poor when enhancer motifs are missing, and that these can be several thousand base-pairs away (25 kb for the heavy chain 3xe2x80x2-enhancer) from the nearest exon.
Mouse models have been established to address the question of the immunogenicity of chimaeric, foreign and authentic antibodies used for therapeutic purposes; see Brxc3xcggemann et al, J. Exp. Med. 170:2153 (1989). It became clear that only authentic proteins escape the surveillance of the immune system.
The techniques currently used for making human antibodies involve either in vitro immunisation and immortalisation of human lymphocytes or genetic engineering. The selection of rare specific antibody-producing human lymphocytes outside the body is difficult and, once the lines are obtained, their yield and stability are poor; see Borrebaeck, Immunol. Today 9:355 (1988). Genetic engineering (also termed xe2x80x9chumanisationxe2x80x9d of rodent antibodies) firstly has to be done for each individual mouse or rat antibody of therapeutic use and secondly does not yield completely human antibodies; see Riechmann et al, Nature 332:323 (1988). xe2x80x9cHumanisingxe2x80x9d existing rodent antibodies already approved for therapy is currently the most successful way to obtain less immunogenic reagents. However, it would be a considerable improvement to have a mouse strain available which makes authentic human antibodies after immunisation with human materials.
A repertoire of immunoglobulins has been obtained from transgenic mice carrying inserted human antibody gene segments in germ line configuration; see WO-A-9004036 and Brxc3xcggemann et al, PNAS 86:6709 (1989). A human mini IgH locus has been constructed with variable region genes (Vs), diversity segments (Ds), joining segments (Js) and the xcexc constant region gene (Cxcexc). The human gene segments rearrange in the lymphoid tissue of these mice (VDJ-Cxcexc) and antibodies with human xcexc heavy chains can be obtained after immunisation. However, the level of human IgM as opposed to mouse IgM is low, and specific hybridomas with human xcexc chains are rare after immunisation. A further complication is that most of those cells that produce human heavy chain also secrete endogenous mouse Ig. This means that rearrangement of human xcexc does not stop endogenous rearrangement; in other words, allelic exclusion is not achieved. Furthermore, the actual repertoire size of the produced human antibodies is unknown but might be small as the IgH construct contains only a limited number of V an: D segments.
Srivastava et al, Gene 103:53-59 (1991), describe plasmids which permit the insertion of neomycin-resistance gene into the human DNA insert or the vector arm of a YAC.
In the latter case, the plasmid also contains a LYS2 gene for selection in a yeast host. The URA3 gene is then replaced by a new insert, thus inactivating URA.
Bothstein et al, Science 240:1439-1443 (1988), describe the practical advantages of the yeast organism for cloning and manipulating large DNA molecules. Green et al, Science 250:94-98 (1990), report the cloning of segments c. us to one million base-pairs in yeast cells in artificial chromosomes (YACs), allowing the long-range mapping and analysis of complex genomes. Yeast vectors for cloning of large DNA molecules combine two features; plasmid sequences for their propagation in E. coli and yeast specific sequences to ensure the replication and maintenance of a linear molecule when grown in yeast.
Nevertheless, the problem of producing large molecules (of which specific examples are immunoglobulins, Factor VIII and Factor IX) on a commercial scale, remains. At the molecular level, it would be desirable to introduce large DNA molecules containing single genes of considerable size, in order to facilitate correct expression. For example, the human Ig heavy chain locus accommodating all Vs, Ds, Js and C regions is estimated to spread over 3000 kb of DNA; the Factor VIII gene is almost 200 kb in size with 23 exons. At present, high level of expression is rarely achieved by the introduction of cDNAs and engineers genomic xe2x80x9cminigenesxe2x80x9d into transgenic animals.
Surprisingly, it has now been found that specific DNA of considerable length can be introduced into ES cells via a YAC, without introducing DNA from the yeast. The need to purify the YAC out of yeast is thus avoided, and transformation is essentially foolproof. This discovery is applicable to the introduction of such DNA into other cells also.
The appropriate YAC, for use in the present invention, includes a foreign gene or gene locus (i.e. including one or more genes or gene segments) of at least 100 kb and also a marker gene which allows selection in cells that are not prokaryotic or yeast cells. This construct can be obtained by integration of the marker gene, e.g. Neo, into a YAC including the foreign gene.
In a further aspect of the invention, ES cells or other cells are transformed by the marked YAC, and thus can be selected and inserted into, say, animals which consequently express human immunoglobulin or other genes. In particular, a transgenic lactating, ovine cr bovine animal, mouse or other rodent, or any non-human animal, may contain a foreign gene or gene locus of considerable length, e.g. at least 100 kb, often at least 300 kb, but also 1 Mb or more, if required. The animal may express the product of another animal, e.g. a human product, but not its own corresponding product.